2-aminopyrimidine derivatives for the treatment of viral infections

ABSTRACT

This invention relates to 2-aminopyrimidine derivatives, processes for their preparation, pharmaceutical compositions, and their use in treating viral infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.15/464,050 filed on Mar. 20, 2017, which is a continuation of U.S.patent application Ser. No. 14/769,773 filed on Aug. 21, 2015, now U.S.Pat. No. 9,598,378, which is a national phase entry of InternationalApplication No. PCT/EP2014/053273, filed on Feb. 20, 2014, which claimspriority to EP Patent Application No. 13156167.2, filed on Feb. 21,2013, each of which is incorporated herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 11, 2017, isnamed TIP0291USCNT2_SL.txt and is 680 bytes in size.

This invention relates to 2-aminopyrimidine derivatives, processes fortheir preparation, pharmaceutical compositions, and their use intreating viral infections.

The present invention relates to the use of 2-aminopyrimidinederivatives in the treatment of viral infections, immune or inflammatorydisorders, whereby the modulation, or agonism, of toll-like-receptors(TLRs) is involved. Toll-Like Receptors are primary transmembraneproteins characterized by an extracellular leucine rich domain and acytoplasmic extension that contains a conserved region. The innateimmune system can recognize pathogen-associated molecular patterns viathese TLRs expressed on the cell surface of certain types of immunecells. Recognition of foreign pathogens activates the production ofcytokines and upregulation of co-stimulatory molecules on phagocytes.This leads to the modulation of T cell behaviour.

It has been estimated that most mammalian species have between ten andfifteen types of Toll-like receptors. Thirteen TLRs (named TLR1 toTLR13) have been identified in humans and mice together, and equivalentforms of many of these have been found in other mammalian species.However, equivalents of certain TLR found in humans are not present inall mammals. For example, a gene coding fora protein analogous to TLR10in humans is present in mice, but appears to have been damaged at somepoint in the past by a retrovirus. On the other hand, mice express TLRs11, 12, and 13, none of which are represented in humans. Other mammalsmay express TLRs which are not found in humans.

Other non-mammalian species may have TLRs distinct from mammals, asdemonstrated by TLR14, which is found in the Takifugu pufferfish. Thismay complicate the process of using experimental animals as models ofhuman innate immunity.

For reviews on TLRs see the following journal articles. Hoffmann, J. A.,Nature, 426, p 33-38, 2003; Akira, S., Takeda, K., and Kaisho, T.,Annual Rev. Immunology, 21, p 335-376, 2003; Ulevitch, R. J., NatureReviews: Immunology, 4, p 512-520, 2004.

Compounds indicating activity on Toll-Like receptors have beenpreviously described such as purine derivatives in WO 2006/117670,adenine derivatives in WO 98/01448 and WO 99/28321, and pyrimidines inWO 2009/067081.

However, there exists a strong need for novel Toll-Like receptormodulators having preferred selectivity, higher potency, highermetabolic stability, and an improved safety profile compared to thecompounds of the prior art.

In accordance with the present invention a compound of formula (I) isprovided

or a pharmaceutically acceptable salt, tautomer(s), stereo-isomericforms, solvate or polymorph thereof, whereinX represents S, S═O or O═S═O,R₁ is hydrogen, (C₁₋₆)-alkyl, (C₁₋₆)-alkoxy or aryl,R₂ is (C₁₋₃)-alkyl or (C₃₋₆)-cycloalkyl andn=1 or 2.

The compounds of formula (I) and their pharmaceutically acceptablesalts, tautomer(s), stereo-isomeric forms, solvate or polymorph thereofhave activity as pharmaceuticals, in particular as modulators ofToll-Like Receptors (especially TLR7 and/or TLR8 activity).

In a further aspect the present invention provides a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt, tautomer, stereo-isomeric form, solvate or polymorphthereof together with one or more pharmaceutically acceptableexcipients, diluents or carriers.

Furthermore a compound of formula (I) or a pharmaceutically acceptablesalt, solvate, tautomer, stereo-isomeric form or polymorph thereofaccording to the current invention, or a pharmaceutical compositioncomprising said compound of formula (I) or a pharmaceutically acceptablesalt, solvate, tautomer, stereo-isomeric form or polymorph thereof canbe used as a medicament.

Another aspect of the invention is that a compound of formula (I) or itspharmaceutically acceptable salt, solvate, tautomer, stereo-isomericform or polymorph thereof, or said pharmaceutical composition comprisingsaid compound of formula (I) or a pharmaceutically acceptable salt,solvate, tautomer, stereo-isomeric form or polymorph thereof can be usedaccordingly in the treatment of a disorder where the modulation ofTLR's, more specifically TLR7 and/or TLR8, is involved.

The term “(C₁₋₆)-alkyl” or “(C₁₋₃)-alkyl refers to a straight-chain,branched-chain or cyclic saturated aliphatic hydrocarbon containing thespecified number of carbon atoms.

The term “aryl” means an aromatic ring structure optionally comprisingone or two heteroatoms selected from N, O and S, in particular from Nand O. Said aromatic ring structure may have 4, 5, 6 or 7 ring atoms. Inparticular, said aromatic ring structure may have 5 or 6 ring atoms.

The term “(C₁₋₆)-alkoxy” refers to an alkyl (carbon and hydrogen chain)group singular bonded to oxygen like for instance a methoxy group orethoxy group.

The term “(C₃₋₆)-cycloalkyl” means refers to a carbocyclic ringcontaining the specified number of carbon atoms.

As used herein, any chemical formula with bonds shown only as solidlines and not as solid wedged or hashed wedged bonds, or otherwiseindicated as having a particular configuration (e.g. R, S) around one ormore atoms, contemplates each possible stereoisomer, or mixture of twoor more stereoisomers.

The terms “stereoisomers”, “stereoisomeric forms” or “stereochemicallyisomeric forms” hereinbefore or hereinafter are used interchangeably.

The invention includes all stereoisomers of the compounds of theinvention either as a pure stereoisomer or as a mixture of two or morestereoisomers. Enantiomers are stereoisomers that are non-superimposablemirror images of each other. A 1:1 mixture of a pair of enantiomers is aracemate or racemic mixture.

Diastereomers (or diastereoisomers) are stereoisomers that are notenantiomers, i.e. they are not related as mirror images. If a compoundcontains a double bond, the substituents may be in the E or the Zconfiguration. If a compound contains an at least disubstitutednon-aromatic cyclic group, the substituents may be in the cis or transconfiguration.

Therefore, the invention includes enantiomers, diastereomers, racemates,E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof,whenever chemically possible.

The meaning of all those terms, i.e. enantiomers, diastereomers,racemates, E isomers, Z isomers, cis isomers, trans isomers and mixturesthereof are known to the skilled person.

The absolute configuration is specified according to theCahn-Ingold-Prelog system. The configuration at an asymmetric atom isspecified by either R or S.

Resolved stereoisomers whose absolute configuration is not known can bedesignated by (+) or (−) depending on the direction in which they rotateplane polarized light. For instance, resolved enantiomers whose absoluteconfiguration is not known can be designated by (+) or (−) depending onthe direction in which they rotate plane polarized light.

When a specific stereoisomer is identified, this means that saidstereoisomer is substantially free, i.e. associated with less than 50%,preferably less than 20%, more preferably less than 10%, even morepreferably less than 5%, in particular less than 2% and most preferablyless than 1%, of the other stereoisomers. Thus, when a compound ofFormula (I) is for instance specified as (R), this means that thecompound is substantially free of the (S) isomer; when a compound ofFormula (I) is for instance specified as E, this means that the compoundis substantially free of the Z isomer; when a compound of Formula (I) isfor instance specified as cis, this means that the compound issubstantially free of the trans isomer.

Pharmaceutically acceptable salts of the compounds of formula (I)include the acid addition and base salts thereof. Suitable acid additionsalts are formed from acids which form non-toxic salts. Suitable basesalts are formed from bases which form non-toxic salts.

The compounds of the invention may also exist in unsolvated and solvatedforms. The term “solvate” is used herein to describe a molecular complexcomprising the compound of the invention and one or morepharmaceutically acceptable solvent molecules, for example, ethanol.

The term “polymorph” refers to the ability of the compound of theinvention to exist in more than one form or crystal structure.

The compounds of the present invention may be administered ascrystalline or amorphous products. They may be obtained for example assolid plugs, powders, or films by methods such as precipitation,crystallization, freeze drying, spray drying, or evaporative drying.They may be administered alone or in combination with one or more othercompounds of the invention or in combination with one or more otherdrugs. Generally, they will be administered as a formulation inassociation with one or more pharmaceutically acceptable excipients. Theterm “excipient” is used herein to describe any ingredient other thanthe compound(s) of the invention. The choice of excipient dependslargely on factors such as the particular mode of administration, theeffect of the excipient on solubility and stability, and the nature ofthe dosage form.

The compounds of the present invention or any subgroup thereof may beformulated into various pharmaceutical forms for administrationpurposes. As appropriate compositions there may be cited allcompositions usually employed for systemically administering drugs. Toprepare the pharmaceutical compositions of this invention, an effectiveamount of the particular compound, optionally in addition salt form, asthe active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirably inunitary dosage form suitable, for example, for oral, rectal, orpercutaneous administration. For example, in preparing the compositionsin oral dosage form, any of the usual pharmaceutical media may beemployed such as, for example, water, glycols, oils, alcohols and thelike in the case of oral liquid preparations such as suspensions,syrups, elixirs, emulsions, and solutions; or solid carriers such asstarches, sugars, kaolin, diluents, lubricants, binders, disintegratingagents and the like in the case of powders, pills, capsules, andtablets. Because of their ease in administration, tablets and capsulesrepresent the most advantageous oral dosage unit forms, in which casesolid pharmaceutical carriers are obviously employed. Also included aresolid form preparations that can be converted, shortly before use, toliquid forms. In the compositions suitable for percutaneousadministration, the carrier optionally comprises a penetration enhancingagent and/or a suitable wetting agent, optionally combined with suitableadditives of any nature in minor proportions, which additives do notintroduce a significant deleterious effect on the skin. Said additivesmay facilitate the administration to the skin and/or may be helpful forpreparing the desired compositions. These compositions may beadministered in various ways, e.g., as a transdermal patch, as aspot-on, as an ointment. The compounds of the present invention may alsobe administered via inhalation or insufflation by means of methods andformulations employed in the art for administration via this way. Thus,in general the compounds of the present invention may be administered tothe lungs in the form of a solution, a suspension or a dry powder.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in unit dosage form for ease ofadministration and uniformity of dosage. Unit dosage form as used hereinrefers to physically discrete units suitable as unitary dosages, eachunit containing a predetermined quantity of active ingredient calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. Examples of such unit dosage forms aretablets (including scored or coated tablets), capsules, pills, powderpackets, wafers, suppositories, injectable solutions or suspensions andthe like, and segregated multiples thereof.

Those of skill in the treatment of infectious diseases will be able todetermine the effective amount from the test results presentedhereinafter. In general it is contemplated that an effective dailyamount would be from 0.01 mg/kg to 50 mg/kg body weight, more preferablyfrom 0.1 mg/kg to 10 mg/kg body weight. It may be appropriate toadminister the required dose as two, three, four or more sub-doses atappropriate intervals throughout the day. Said sub-doses may beformulated as unit dosage forms, for example, containing 1 to 1000 mg,and in particular 5 to 200 mg of active ingredient per unit dosage form.

The exact dosage and frequency of administration depends on theparticular compound of formula (I) used, the particular condition beingtreated, the severity of the condition being treated, the age, weightand general physical condition of the particular patient as well asother medication the individual may be taking, as is well known to thoseskilled in the art. Furthermore, it is evident that the effective amountmay be lowered or increased depending on the response of the treatedsubject and/or depending on the evaluation of the physician prescribingthe compounds of the instant invention. The effective amount rangesmentioned above are therefore only guidelines and are not intended tolimit the scope or use of the invention to any extent.

Preparation of Compounds of Formula (I)

Overall scheme. Compound A was prepared according to proceduresdescribed in WO2008147697 and WO2009067081.

EXPERIMENTAL SECTION

Triethylamine (10.5 g, 103.75 mmol, 2.4 eq.) was added to the solutionof A (10 g, 43.23 mmol, 1 eq.) in CH₂Cl₂ (200 mL) at 0° C.Methanesulfonyl chloride (6.4 g, 55.87 mmol, 1.3 eq.) was added dropwiseto the solution and stirred 1.5 hours at 0° C. CH₂Cl₂ (500 mL) wasadded. The solution was washed with aq. NaHCO₃, brine, and dried overNa₂SO₄, the solids were removed by filtration and the solvent of thefiltrate was removed under reduced pressure to give B. Used as suchwithout further purification.

A mixture of B (12 g, 38.782 mmol, 1 eq.) and sodium thiomethoxide (4.08g, 58.17 mmol, 1.5 eq.) in DMF (60 mL) was stirred overnight at 70° C.The solids were removed by filtration and the solvents of the filtratewere removed under reduced pressure. The crude was dissolved in ethylacetate, washed with water, brine, dried over Na₂SO₄, the solids wereremoved by filtration and the solvents of the filtrate were removedunder reduced pressure. The crude was purified by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate from 40/1 to 3/1)to afford C.

¹H NMR (400 MHz, chloroform-d) δ ppm 0.70-0.85 (m, 5H), 1.15-1.49 (m,13H), 1.49-1.61 (m, 1H), 1.61-1.80 (m, 1H), 2.05 (s, 3H), 2.38-2.50 (m,2H), 3.51 (br. s., 1H), 4.25 (br. s., 1H)

HCl/dioxane (47 mL, 187.43 mmol, 10 eq.) was added drop wise to astirred solution of C (4.9 g, 18.74 mmol, 1 eq.) in CH₂Cl₂ at 0° C., andstirred for 1 hour at 25° C. The solution was concentrated under reducedpressure to give D. Used as such in the next step.

D (0.75 g, 3.79 mmol, 1 eq.), 2-amino-4-chloro-5-methoxypyrimidine(0.908 g, 5.69 mmol, 1.5 eq.) and K₂CO₃ (1.57 g 11.38 mmol, 3 eq.) weremixed in ethanol (20 mL). The mixture was stirred at 120° C. in themicrowave for 30 minutes. The solvent was removed under reducedpressure. The crude was purified by preparative silica thin layerchromatography (eluent: CH₂Cl₂:CH₃OH=20:1) to afford E.

¹H NMR (400 MHz, chloroform-d) δ ppm 1.05-1.15 (m, 3H), 1.40-1.60 (m,4H), 1.95 (m, 2H), 2.15 (m, 1H), 2.30 (d, 3H), 2.70 (t, 1H), 2.90 (t,1H), 3.55 (m, 1H), 4.50 (m, 1H), 3.95 (s, 3H), 6.20 (d, 1H), 6.60 (br.s., 2H), 7.45 (s, 1H)

Oxone (6.959 g, 11.32 mmol, 3 eq.) was added to a solution of E (1.45 g,3.773 mmol, 1 eq.) in DMF (100 mL) and water (100 mL). The mixture wasstirred for 12 hours at 20° C. The solids were removed by filtration andthe filtrate was basified to pH=8 with saturated, aq. Na₂CO₃ solution.The resultant mixture was concentrated under reduced pressure. Theresidue was purified by preparative high-performance liquidchromatography (column: gemini 150×30 mm×5μm, C18, mobile phase:CH₃CN/water (0.05% HCl), Gradient: 2-32% CH₃CN, 0-8 min, flow rate: 30mL/min). The best fractions were pooled and concentrated under reducedpressure to afford 1.

LC-MS 3.88 min

¹H NMR (400 MHz, methanol-d₄) δ ppm 0.92 (t, J=6.9 Hz, 3H), 1.21-1.50(m, 4H), 1.69 (q, J=7.1 Hz, 2H), 1.95-2.28 (m, 2H), 2.98 (s, 3H),3.09-3.22 (m, 2H), 3.87 (s, 3H), 4.37-4.55 (m, 1H), 7.26 (s, 1H) labileprotons not observed.

A solution of E (40 mg, 0.14 mmol, 1 eq.) in ethanol (40 mL) was treatedwith a solution of NalO₄ (0.2 g, 1 mmol, 7.5 eq.) in water (10 mL), andthen stirred at room temperature overnight. The solution wasconcentrated under vacuum. The residue was diluted with water andextracted with ethyl acetate. The combined organic layers were washedwith brine, dried over Na₂SO₄, the solids were removed via filtration,and the solvent of the filtrate was removed under reduced pressure. Thecrude was purified by preparative high-performance liquid chromatography(C18 column, eluent: CH₃CN, H₂O from 3/97 to 33/67, 0.05% HCl). Thedesired fractions were collected and concentrated under vacuum to afford2.

LC-MS 3.78 min

¹H NMR (400 MHz, methanol-d₄) δ ppm 0.90 (t, J=6.8 Hz, 3H), 1.19-1.49(m, 4H), 1.67 (d, J=6.5 Hz, 2H), 1.91-2.15 (m, 2H), 2.63 (br. s., 3H),2.69-2.96 (m, 2H), 3.85 (s, 3H), 4.46 (br. s., 1H), 7.25 (s, 1H) labileprotons not observed.

LC-MS Analytical Method.

Column YMC-PACK ODS-AQ, 50 × 2.0 mm, 5 μm Mobile Phase A: H₂O (0.1% TFA)B: acetonitrile (0.05% TFA) TIME(min) A % B % 0 100 0 1 100 0 5 40 607.5 40 60 8 100 0 Flow Rate 0.8 mL/min Wavelength UV 220 nm ColumnTemperture 50° C. MS polarity positive LCMS Agilent 1100

Biological Activity of Compounds of Formula (I) Description ofBiological Assays Assessment of TLR7 and TLR8 Activity

The ability of compounds to activate human TLR7 and/or TLR8 was assessedin a cellular reporter assay using HEK293 cells transiently transfectedwith a TLR7 or TLR8 expression vector and NFκB-luc reporter construct.

Briefly, HEK293 cells were grown in culture medium (DMEM supplementedwith 10% FCS and 2 mM Glutamine). For transfection of cells in 15 cmdishes, cells were detached with Trypsin-EDTA, transfected with a mix ofCMV-TLR7 or TLR8 plasmid (1700 ng), NFκB-luc plasmid (850 ng) and atransfection reagent and incubated for 48 h at 37° C. in a humidified 5%CO₂ atmosphere. Transfected cells were then washed in PBS, detached withTrypsin-EDTA and resuspended in medium to a density of 1.25×10⁵cells/mL. Forty microliters of cells were then dispensed into each wellin 384-well plates, where 200 nL of compound in 100% DMSO was alreadypresent. Following 6 hours incubation at 37° C., 5% CO₂, the luciferaseactivity was determined by adding 15 μL of Steady Lite Plus substrate(Perkin Elmer) to each well and readout performed on a ViewLux ultraHTSmicroplate imager (Perkin Elmer). Dose response curves were generatedfrom measurements performed in quadruplicates. Lowest effectiveconcentrations (LEC) values, defined as the concentration that inducesan effect which is at least two fold above the standard deviation of theassay, were determined for each compound.

Compound toxicity was determined in parallel using a similar dilutionseries of compound with 40 μL per well of cells transfected with theCMV-TLR7 construct alone (1.25×10⁵ cells/mL), in 384-well plates. Cellviability was measured after 6 hours incubation at 37° C., 5% CO₂ byadding 15 μL of ATP lite (Perkin Elmer) per well and reading on aViewLux ultraHTS microplate imager (Perkin Elmer). Data was reported as0050.

In parallel, a similar dilution series of compound was used (200 nL ofcompound in 100% DMSO) with 40 μL per well of cells transfected withNFκB-luc reporter construct alone (1.25×10⁵ cells/mL). Six hours afterincubation at 37° C., 5% CO₂, the luciferase activity was determined byadding 15 μl of Steady Lite Plus substrate (Perkin Elmer) to each welland readout performed on a ViewLux ultraHTS microplate imager (PerkinElmer). Counterscreen data is reported as LEC.

Activation of ISRE Promoter Elements

The potential of compounds to induce IFN-I was also evaluated bymeasuring the activation of interferon-stimulated responsive elements(ISRE) by conditioned media from PBMC. The ISRE element of sequenceGAAACTGAAACT (SEQ ID NO: 1) is highly responsive to the STAT1-STAT2-IRF9transcription factor, activated upon binding of IFN-I to their receptorIFNAR (Clontech, PT3372-5W). The plasmid pISRE-Luc from Clontech (ref.631913) contains 5 copies of this ISRE element, followed by the fireflyluciferase ORF. A HEK293 cell line stably transfected with pISRE-Luc(HEK-ISREluc) was established to profile the conditioned PBMC cellculture media.

Briefly, PBMCs were prepared from buffy coats of at least two donorsusing a standard Ficoll centrifugation protocol. Isolated PBMCs wereresuspended in RPMI medium supplemented with 10% human AB serum and2×10⁵ cells/well were dispensed into 384-well plates containingcompounds (70 μL total volume). After overnight incubation, 10 μL ofsupernatant was transferred to 384-well plates containing 5×10³HEK-ISREluc cells/well in 30 μL (plated the day before). Following 24hours of incubation, activation of the ISRE elements was measured byassaying luciferase activity using 40 μL/well Steady Lite Plus substrate(Perkin Elmer) and measured with ViewLux ultraHTS microplate imager(Perkin Elmer). The stimulating activity of each compound on theHEK-ISREluc cells was reported as LEC value, defined as the compoundconcentration applied to the PBMCs resulting in a luciferase activity atleast two fold above the standard deviation of the assay. The LEC inturn indicates the degree of ISRE activation on transfer of a definedamount of PBMC culture medium. Recombinant interferon α-2a (Roferon-A)was used as a standard control compound.

TABLE I BIOLOGICAL ACTIVITY. Human TLR 7 Human TLR 8 HEK-ISRE luc #(LEC) μM (LEC) μM (LEC) μM 1 2.0 1.7 0.65 2 1.4 9.2 4.8 E 3.9 10 NA NA =not available. All compounds showed no toxicity up to the highest testedconcentration. All compounds showed no activity (LEC > 25 μM) in the HEK293 NF-kB counterscreen assay described above.

1. A compound of formula (I)

or a pharmaceutically acceptable salt, tautomer(s), stereo-isomericforms, solvate or polymorph thereof, wherein X represents S, S═O orO═S═O, R₁ is hydrogen, (C₁₋₆)-alkyl, (C₁₋₆)-alkoxy or aryl, R₂ is(C₁₋₃)-alkyl or (C₃₋₆)-cycloalkyl and n=1 or
 2. 2. A pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt, tautomer(s), stereo-isomeric forms, solvate orpolymorph thereof according to claim 1 together with one or morepharmaceutically acceptable excipients, diluents or carriers. 3.(canceled)
 4. (canceled)